1. Field of the Invention
The present invention relates to nucleic acid constructs for expressing an effector gene, methods of making such constructs, and methods of using such constructs.
2. Description of Related Art
A problem in gene therapy which is to a large extent inadequately solved is that of controlling the expression of an effector gene in a cell-specific manner, especially in diseased cells or cells which have otherwise been altered. The present invention comprises a novel process for achieving this control. The present invention is based on the finding (Werness et al., Science 248, 76 (1990)) that, in degenerate cells, regulatory proteins appear which are altered or diminished in such a way that they either are no longer able to bind to their affiliated partner molecules and interact with them, or they gain new binding properties with their affiliated partner molecules or with other partner molecules.
The novel process is furthermore based on the finding that the retinoblastoma protein, for example, is able to bind to the activation domain of the E2F transcription factor and thereby inhibit its activity (Flemington et al., PNAS USA 90, 69 14 (1993)).
Genes for regulatory proteins of this nature have already been used for expression systems for searching for inhibitors or stimulators of these regulatory proteins (e.g. WO95/19367, WO95/14777, WO97/04092).
In addition, vector systems with a first vector expressing a tumor suppressor protein and a second vector expressing a protein which binds to the tumor suppressor protein and thereby inhibits it, have already been disclosed (WO 95/16771). Both the vectors are introduced into one cell. By combining the two vectors, vectors encoding a tumor suppressor protein can be produced in the cell without the proliferation of the cell being inhibited by the tumor suppressor protein.
In addition, WO 97/12970 discloses expression systems in which the expression of a first gene is controlled by a first promoter whose function is suppressed in non-tumor cells, and the expression of a second gene, whose expression product inhibits the expression of the first gene in non-tumor cells, is controlled by a second promoter which is upregulated in non-tumor cells.
The present invention relates to a novel and simple expression system which can only be activated in cells in which such regulatory proteins occur in diminished or altered form. When activated, an effector gene which is encoded by the expression system is transcribed. The expression product of the effector gene has a prophylactic or therapeutic effect, either on its own or in combination with a further pharmaceutically active compound.
The present invention relates to a nucleic acid construct for expressing an effector gene. This nucleic acid construct comprises (a) a first promoter, (b) a transcription factor gene, the expression of which is controlled by the first promoter, (c) a second promoter, to which the gene product of the transcription factor gene binds, and (d) an effector gene, the expression of which is controlled by the second promoter, wherein the activity of the transcription factor gene product depends on one or more cellular regulatory proteins that bind to the transcription factor gene product and affect the activity thereof.
In one embodiment, the nucleic acid construct comprises (a) a first promoter comprising an activation sequence for the transcription of the transcription factor gene, (b) a transcription factor gene comprising (i) an activation domain, (ii) a binding sequence for a cellular regulatory protein, and (iii) a DNA-binding domain; (c) a second promoter comprising an activation sequence which is activated by binding the expression product of the transcription factor gene and activates the transcription of the effector gene, and (d) an effector gene.
In another embodiment of the invention, the nucleic acid construct comprises (a) a first promoter comprising an activation sequence for the transcription of the transcription factor gene comprising (i) a DNA-binding sequence for a cellular regulatory protein and (ii) a basal promoter; (b) a transcription factor gene comprising a gene encoding a repressor protein that inhibits the second promoter; (c) a second promoter comprising (i) an activation sequence for the transcription of the effector gene and (ii) a DNA sequence which binds the repressor protein and thereby inhibits the activation of the transcription of the effector gene; and (d) an effector gene.
The present invention also relates to vectors and isolated cells comprising the aforementioned nucleic acid construct. The vector may be a plasmid vector or a viral vector. The cell may be any cell, including mammalian cells.
The invention further relates to method of making the aforementioned nucleic acid construct comprising ligating the first promoter, transcription factor gene, second promoter, and effector gene together stepwise.
Still further, the present invention relates to methods of treating and/or preventing disease comprising administering to a patient the aforementioned nucleic acid construct or isolated cells containing the aforementioned nucleic acid construct. According to one aspect of the invention, the nucleic acid is administered to a patient externally, perorally, intravesically, nasally, intrabronchially or into the gastrointestinal tract, or injected into an organ, into a body cavity, into the musculature, subcutaneously or into the blood circulation. According to another aspect of the invention, the disease treated or prevented may be infections, tumors, leukemias, autoimmune diseases, allergies, arthritides, inflammations, organ rejections, graft versus host reactions, blood coagulation diseases, circulatory diseases, anemia, hormone diseases and CNS damage.